Western blot analysis of wild type GST-cdc25C and mutant GST-cdc25C Ser216Ala fusion proteins, untreated or phosphorylated by Chk2, using Phospho-cdc25C (Ser216) Antibody.
Western blot analysis of GST-cdc25C phosphorylated by cdc2/cyclin B (New England Biolabs #P6020), Chk2 or both kinases, using Phospho-cdc25C (Ser216) Antibody (upper) or Phospho-cdc25C (Ser214) antibody (lower).
Western Blot analysis of HT29 cells, untreated, nocodazole-treated, and lambda phosphotase-treated, using Phospho-cdc25C (Ser216) antibody (upper), and cdc25C (5H9) Rabbit mAb, #4688, (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-cdc25C (Ser216) Antibody detects endogenous levels of cdc25C only when phosphorylated at Ser216.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser216 of human cdc25C. Antibodies are purified by protein A and peptide affinity chromatography.
Cdc25 is a protein phosphatase responsible for dephosphorylating and activating cdc2, a crucial step in regulating the entry of all eukaryotic cells into mitosis (1). cdc25C is constitutively phosphorylated at Ser216 throughout interphase by c-TAK1, while phosphorylation at this site is DNA damage-dependent at the G2/M checkpoint (2). When phosphorylated at Ser216, cdc25C binds to members of the 14-3-3 family of proteins, sequestering cdc25C in the cytoplasm and thereby preventing premature mitosis (3). The checkpoint kinases Chk1 and Chk2 phosphorylate cdc25C at Ser216 in response to DNA damage (4,5).
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